Method of detecting PS2V

ABSTRACT

A method of detecting PS2V characterized by comprising reacting PS2V in a sample with a primary antibody which is bonded to a solid phase and specifically recognizes PA2V, then reacting with a secondary antibody recognizing PS2 or PS2V by any of the following procedures: (a) reacting with a secondary antibody having been enzyme-labeled and recognizing PS2 or PS2V; (b) reacting with a secondary antibody having been biotinylated and recognizing PS2 or PS2V and then reacting with an avidinylated or streptoavidinylated enzyme; (c) reacting with a secondary antibody having been biotinylated and recognizing PS2 or PS2V and then reacting with a biotinylated enzyme and avidin or streptoavidin; and (d) reacting with a secondary antibody recognizing PS2 or PS2V and then reacting with an antibody having been enzyme-labeled and recognizing the secondary antibody; then adding the substrate of the above enzyme and detecting the product formed by the enzyme reaction.

TECHNICAL FIELD

The present invention relates to a method of detecting PS2V which isrecognized in sporadic Alzheimer's disease by using antigen-antibodyreaction.

BACKGROUND ART

With elderly population progressively increasing, a rapid growth ofdemential patients has become a serious social problem. Alzheimer'sdisease (AD) is a kind of dementia, and account for about 30% of seniledementia in Japan and more than half in Europe and U.S. AD is a kind ofneurodegenerative disease, and its pathological characters include: (1)senile plaques in which A β is accumulated as a principle component areobserved between neuronal cells; (2) abnormal phosphorylated tau proteinaggregates in neuronal cells and fibrotic neurofibril are observed; (3)the cerebrum shrinks (deciduation of cerebral neocortex and neuronalcells of hippocampal). As the clinical characters, AD is a progressivedementia presenting hypergasia in general cognition, notably disorder ofmemory.

AD is classified into familial Alzheimer's disease (FAD), which is smallin the number of cases and shows autosomal dominant inheritance, andsporadic Alzheimer's disease (SAD), which apparently lacks familymedical history and accounts for 90% of the total Alzheimer's disease.As genes causative of FAD, are identified amyloid precursor protein(APP) gene located on chromosome 21, presenilin-1 (PS1) gene found onchromosome 14 and presenilin-2 (PS2) gene located on chromosome 1. Themechanism of development of FAD has been gradually becoming clear. Onthe other hand, SAD which makes up a majority of AD cases presents thesame neuropathological observations as FAD, but its developmentmechanism is unknown in a considerable number of aspects.

For common clinical diagnosis of AD, cognition tests such as SM-IV,NINCDS-ADRDA and the like which are proposed in the United State areutilized. However, it is difficult to diagnose extremely slightcognition impediment in an early stage of AD as dementia usingconventional cognition tests. The currently used definite diagnosis ofAD consists of recognizing deposition of amyloid protein (senileplaques) and accumulation of tau protein (neurofibrillary tangles) inpostmortem brain. Thus effective antemortem early diagnostic methodshave not been established. In the present circumstances, it is too latewhen typical symptoms of AD (specific demential symptoms such asincapability of cognition and the like) are recognized.

At present, several kinds of anti-dementia medicines are distributed ina lot of countries, and donepezil has been clinically used in Japansince 1999. These medicines often have a beneficial effect onearly-stage cases. Since the medicines are expected to exhibitbeneficial effect if AD is diagnosed early, there is a demand fordevelopment of a diagnostic marker effective for early treatment of AD.

It has been reported that mRNA of a splicing variant (PS2V) of a PS2gene with deletion of its fifth exon are observed manifesting frequentlyin about 70% of the encephala of SAD patients (J. Neurochem., Vol. 72,No. 6, 1999, 2498-2505). The mRNA of PS2V codes for a protein consistingof 124 amino acids having 5 amino acids (Ser-Ser-Met-Ala-Gly) (SEQ IDNO:7) added to 119 amino acid residues (Met1 to Leu119) at the Ntenninal of PS2.

Immunohistologic detection of PS2V using samples of CA1 regions of thehippocampi of the encephala of SAD patients have confirmed 100%manifestation of PS2V (J. Biol Chem, 2001 Jan. 19; 276(3):2108-2114).

In vitro analysis has shown that {circle around (1)} in humanneuroblastoma SK-N-SH cells in which PS2V is forced to expressed,susceptibility to endoplasmic reticulum (ER) stress increases since theinduction of stress responsive protein GRP78 is suppressed; that {circlearound (2)} PS2V inhibits autophosphorylation of Ire1 protein (ER stresssensor) and inactivates ER stress response, thereby causing thesuppression on the expression of GRP78; and that {circle around (3)} incells expressing PS2V, the production of both Aβ1-40 and Aβ1-42 isincreased. From these three points, it is considered that in SAD, theexpression of PS2V may possibly trigger neuronal death and increasing ofAβ production.

Accordingly, it is considered that highly sensitive detection of PS2Vleads to early diagnosis of AD since the expression of PS2V plays animportant role in AD development.

As the detection of PS2V, mention may be made of the detection of PS2Vitself and the detection of mRNA of PS2V. For early diagnosis, it isimpossible to collect a brain tissue of the encephalon of a patient.Therefore, it is necessary to carry out a test using a body fluid of apatient such as cerebrospinal fluid, blood, serum, urine or the likewhich can be collected relatively easily. However, mRNA is retained incerebrospinal fluid or serum only for such an extremely short time andis decomposed so quickly that the diagnosis by detecting mRNA isimpossible. On the other hand, PS2V is retained in cerebrospinal fluidor serum for a long time as compared with mRNA, and therefore, it isconsidered suitable for the diagnosis. However, since only a traceamount of PS2V is present in cerebrospinal fluid or serum, a method ofdetecting PS2V with high sensitivity is required.

Japanese Unexamined Patent Application Publication NO. 2000-37192 hasalready disclosed a method of producing PS2V by inducing the expressionof an abnormal splicing varient of the PS2 gene using oxidative stressloading and β-amyloid stimulation in a culture system of neuronal cells.However, highly sensitive detection of PS2V has not been found.

Conventionally, alkaline phosphatase (referred to as ALP hereinafter) isoften used as a labeling molecule in enzyme immunoassay and nucleic aciddetection. Since ALP hydrolyzes a substrate which is a phosphate ester,the quantity of an object biomolecule conjugated with ALP can bedetermined by determining the quantity of a product obtained byhydrolysis with ALP.

Depending upon different determination techniques for the product, thedetection of the product is classified into four types, i.e.,absorptiometric detection, chemoluminescent detection, fluorescentdetection and electrochemical detection.

Literature such as Analytica Chemica Acta 393 (1999) 95-102 and othershave reported electrochemical detection using p-methoxyphenyl phosphoricacid as a substrate for ALP. However, methods of highly sensitivedetection of PS2V using these substrates have not been found.

Accordingly, there are desired a method of highly sensitive detection ofPS2V which is applicable to early diagnosis of AD and a substrate for aconjugating enzyme usable for the method.

DISCLOSURE OF INVENTION

The present invention provides a method of detecting PS2V characterizedby comprising:

reacting PS2V in a sample with a primary antibody which is immobilizedto a solid phase,

executing any one of the following steps (a) to (d):

-   -   (a) reacting the PS2V with a secondary antibody which is marked        with a label;    -   (b) reacting the PS2V with a biotinylated secondary antibody,        followed by reaction with an avidinated or streptoavidinated        label;    -   (c) reacting the PS2V with a biotinylated secondary antibody,        followed by reaction with a biotinylated label and avidin or        streptoavidin; and    -   (d) reacting the PS2V with a secondary antibody, followed by        reaction with an antibody which is marked with a label and        recognizes the secondary antibody; and

subsequently, determining the amount of the label captured by the solidstate.

Here, the combination of the primary and secondary antibodies may beeither one of the following sets (a) and (b).

-   (a) The primary antibody which specifically recognizes PS2V and the    secondary antibody which recognizes PS2 or PS2V.-   (b) The primary antibody which recognizes PS2 or PS2V and the    secondary antibody which specifically recognizes PS2V.

When a label is an enzyme, suitably used is a method of detecting PS2Vcharacterized by comprising:

reacting PS2V in a sample with a primary antibody which is immobilizedto a solid phase and specifically recognizes PS2V,

executing any one of the following steps (a) to (d):

-   -   (a) reacting the PS2V with a secondary antibody which is labeled        with an enzyme and recognizes PS2 or PS2V;    -   (b) reacting the PS2V with a biotinylated secondary antibody        which recognizes PS2 or PS2V, followed by reaction with an        avidinated or steptoavidinated enzyme;    -   (c) reacting the PS2V with a biotinylated secondary antibody        which recognizes PS2 or PS2V, followed by reaction with a        biotinylated enzyme and avidin or streptoavidin; and    -   (d) reacting the PS2V with a secondary antibody which recognizes        PS2 or PS2V, followed by the reaction with an antibody which is        conjugated with an enzyme and recognizes the secondary antibody;        and

subsequently, adding a substrate for the enzyme and detecting a productgenerated by enzymatic reaction.

Alternatively, the present invention provides a method of detecting PS2Vcharacterized by comprising:

executing any one of the following steps (a) to (d) in the presence ofPS2V derived from a sample and a certain amount of PS2V immobilized to asolid state;

-   -   (a) reacting PS2V with a primary antibody which is marked with a        label and specifically recognizes PS2V;    -   (b) reacting PS2V with a biotinylated primary antibody which        specifically recognizes PS2V, followed by reaction with an        avidinated or streptoavidinated label;    -   (c) reacting PS2V with a biotinylated primary antibody which        specifically recognize PS2V, followed by reaction with a        biotinylated label and avidin or streptoavidin; and    -   (d) reacting PS2V with a primary antibody which specifically        recognizes PS2V, followed by reaction with a secondary antibody        which is marked with a label and recognizes the primary        antibody; and

subsequently determining the amount of the label captured by the solidstate and detecting the PS2V derived from the sample by calculation fromthe certain amount of the primary antibody and the determined amount ofthe label.

The present invention further provides a method of detecting PS2Vcharacterized by comprising:

-   -   reacting a certain amount of an antibody which is immobilized to        a solid phase and specifically recognizes PS2V with PS2V derived        from a sample and a certain amount of PS2V marked with a label        in competition against each other;

determining the amount of the label captured by the solid phase; and

detecting the PS2V derived from the sample by calculation from thecertain amount of the antibody and the determined amount of the label.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the result of Example 1 of the present invention in whichchemoluminescent was determined by sandwich method using rGST-PS2V as asample;

FIG. 2 shows the result of the present invention of determination ofPS2V in cerebrospinal fluid of patients of subarachnoid hemorrhage,hydrocephalia and sporadic Alzheimer's disease;

FIG. 3 shows the result of Example 1 of the present invention in whichabsorbance was determined by sandwich method using rGST-PS2V as asample;

FIG. 4 shows the construction of FIA-EC-DT used in electrochemicaldetection according to the present invention;

FIG. 5 shows the construction of an FIA-EC-DT detector according to thepresent invention;

FIG. 6 shows the result of Example 2 of the present invention in whichelectrochemical detection was carried out by sandwich method usingrGST-PS2V as a sample (dependency upon the amount of an antigen, anaverage value of the detection repeated three times);

FIG. 7 shows the result of Example 2 of the present invention in whichelectrochemical detection was carried out by sandwich method usingrGST-PS2V as a sample (values are obtained by deducting a backgroundvalue obtained with no antigen used);

FIG. 8 shows the result of Example 3 of the present invention in whichabsorbance was determined by competitive assay using rGST-PS2V as asample;

FIG. 9 shows the results of Example 4 of the present invention in whichelectrochemical detection was carried out by competitive assay usingrGST-PS2V as a sample.

BEST MODE FOR CARRYING OUT THE INVENTION

PS2V (SEQ.ID.NO. 1) to be detected according to the present invention isa polypeptide derived from a splicing variant of presenilin-2 whichlacks exon 5. PS2V has an amino acid sequence represented SEQ ID NO: 2or an amino acid sequence including addition, deletion or substitutionof one to several amino acids with respect to the amino acid sequence ofSEQ ID NO: 2. The number of amino acids which may be added, deleted orsubstituted is generally 1 to about 20, preferably 1 to about 10, andmore preferably 1 to about 5. Such a polypeptide generally has about 80%or more, preferably 90% or more, more preferably 95% or more homologywith the amino acid sequence of SEQ ID NO:2.

The normal PS2 gene has a cDNA sequence represented by SEQ ID NO: 3, andthe cDNA sequence corresponding to the splicing variant (PS2V) genelacking exon 5 (exons 4 and 6 are connected) is shown in SEQ.ID.NO. 4.In the base sequence of SEQ ID NO: 4, the junction site of exon 4 withexon 6 lies between bases 705th and 706th. The base sequence of SEQ IDNO: 1 corresponds to bases 359th to 724th of SEQ.ID.NO. 4. SEQ ID NOs: 1to 4 are disclosed in Japanese Unexamined Patent Publication No.2000-37192.

Samples used according to the present invention may preferably be bodyfluid samples such as cerebrospinal fluid, serum and the like.

The antibody which specifically recognizes PS2V means an antibody whichdoes not recognize PS2 but recognizes PS2V. The antibody recognizes aregion including the amino acid sequence of Ser-Ser-Met-Ala-Gly at theC-terminal of the amino acid sequence of SEQ ID NO: 2.

The antibody which recognizes PS2 or PS2V means an antibody whichrecognizes an amino acid sequence common to PS2 and PS2V, for example,an antibody which recognizes amino acid residues 42nd to 58th of theamino acid sequence of PS2.

These antibodies can be obtained by preparing a peptide includingSer-Ser-Met-Ala-Gly (SEQ ID NO:7) and a peptide including amino acidresidues 42nd to 58th of the amino acid sequence of PS2, immunizingrabbits or the like with these peptides for a certain period of time andcollecting serum from the rabbits according to the conventional method.

These antibodies are preferably used in the form of F(ab′)₂ or Fab′fragments for suppressing non-specific reaction.

For labeling antibody, a radioisotope (¹²⁵I), the enzymes, fluorescentand luminous molecule (a dye such as fluoresceine, fluorescent proteinsuch as GFP, a complex coordinated to rare earth element, material oforganic EL element, and so on), chromophore (photoactive yellow protein:PYP, and so on), and electroactive species (ferrocene, and so on) may beused.

For labeling an antibody with an enzyme, the antibody may be labeleddirectly with the enzyme to form an enzyme-labeled antibody. Or theantibody may be biotinylated, and then an avidinated (orstreptoavidinated) enzyme may be bound to the biotinylated antibodytaking advantage of specific bonding of biotin and avidin(streptoavidin). Or the biotinylated antibody may be reacted with avidin(or streptoavidin) and a biotinylated enzyme. Or another antibody whichrecognizes the antibody and is labeled with the enzyme may be bound tothe unlabeled antibody.

In the case where PS2V needs to be detected with high sensitivity, thereoften arise problems of decline in the activity of the antibody causedby labeling reaction and non-specific adsorption of the enzyme-labeledantibody. Thus, it is important to suppress modification to anantigen-binding site of the antibody and preventing multiplicity of theantibody. For this purpose, the enzyme may be introduced to thiol groupof Fab′. More particularly, instead of using a reagent which modifiesamino group of the antibody unselectively, a bifunctionalhetero-crosslinking agent such as N-succinimidyl-6-maleimidohexanoatemay be used to make a composite between amino group of an enzyme andthiol group obtained by reduction of disulfide of the antibody. Furthermore particularly, IgG is digested with pepsin to remove a highlyhydrophobic Fc region and F(ab′)₂ fragment is collected. The disulfidebonding at a hinge portion is reduced under moderate conditions using 2mmol/L solution of 2-mercaptoethylamine (0.1 mol/L sodium phosphatebuffer containing 5 mmol/L EDTA, pH6.0, 37° C., 90 minutes) to formthiol group, thereby Fab′ being prepared. After concentration of Fab′,the enzyme-labeled antibody is obtained by reaction with an enzyme towhich maleimido group is introduced.

A known enzyme available for labeling can be used as the labelingenzyme. Examples of such enzymes include HRP (horseradish peroxidase),ALP (alkaline phosphatase), β-D-galactosidase, glucose-6-phosphatedehydrogenase, luciferase and the like. A substrate for the enzyme maybe selected from light-absorptive substrates, fluorescent substrates andluminous substrates as appropriate according to the enzyme to be used.Examples of such substrates include luminol, o-phenylenediamine(OPD),3,3′,5,5′-tetramethylbenzidine (TMB), 2-nitrophenyl-β-D-galactoside,4-nitrophenyl-phosphate, 4-methylumbelliferyl-β-D-galactoside,glucose-6-phosphate, 4-hydroxyphenylacetic acid,3-(4-hydroxyphenyl)propionic acid and the like. In the case where theenzyme is ALP, the substrate therefor may be p-nitrophenyl phosphate(pNPP), phenyl phosphoric acid, aminophenyl phosphate, naphthylphosphate, 4-methylbelliferyl phosphate, and the like.

Especially in the case where the enzyme is ALP in the present invention,the substrate may preferably be a phosphate ester of a phenol derivativehaving at least an electron donating group at o-position and/or atp-position, more preferably p-methoxyphenyl phosphate. Examples of theelectron donating group include an alkyl, an ether, a silyl and an aminogroup, and derivatives thereof. Preferably, the electron donating groupmay be a lower alkoxy group. Further more, using p-methoxyphenylphosphate is more preferable.

The method of detecting PS2V of the present invention will be describedbelow by giving an example in the case where an antibody whichspecifically recognizes PS2V is used as the primary antibody, anantibody which recognizes PS2 or PS2V is used as the secondary antibody,and an enzyme is used as a label. In the above description, themeasurement may also be carried out in the case that the primary andsecondary antibodies are exchanged.

First, the primary antibody which does not react with PS2 but reactswith PS2V (which specifically recognizes PS2V) is immobilized to a solidphase. As the solid phase, may be used a test tube, a 96-well plate,beads and the like. The primary antibody can be immobilized to the solidphase by a known method, for example, by physical adsorption and bychemical binding using a crosslinking agent such as glutaraldehyde,carbodiimide and the like.

After the antibody is immobilized, the solid phase is blocked with useof a blocking agent such as BSA (bovine serum albumin), casein, PVA(polyvinyl alcohol), skim milk, keratin, dextran, gelatin and the like,for preventing non-specific adsorption to the solid phase.

Next, PS2V in a sample is reacted with the primary antibody immobilizedto the solid phase at a reaction temperature of about 4 to 40° C.,preferably about 4 to 30° C. for a reaction time of about 5 minutes toovernight, preferably for about 40 minutes to 6 hours. By this step,only PS2V is bound to the primary antibody to be captured by the solidphase.

After B/F separation, the resulting PS2V is reacted with the secondaryantibody which recognizes PS2 or PS2V. The reaction conditions are thesame as those for the primary antibody. The secondary antibody may beconjugated directly with an enzyme as described above. Or thebiotinylated labeled antibody may also be reacted with an avidinated (orstreptoavidinated) enzyme to form an enzyme-labeled antibody.

The biotinylated secondary antibody may also be reacted with avidin (orstreptoavidin) and a biotinylated enzyme to form an enzyme-conjugatedantibody. Alternatively, the PS2V is reacted with the secondaryantibody, unlabeled, and then with an enzyme-labeled antibody whichrecognizes the secondary antibody, which may be allowed to react forenzyme-labeling.

Next, a substrate for enzyme is added and allowed to react with theenzyme. Then, a product generated by enzymatic reaction is detected.Suitable reaction conditions including adequate reaction temperature andappropriate reaction time may be varied depending upon a combination ofan enzyme and a substrate to be used. For example, the cases where HRPis used as the enzyme are explained as follows. (1) If OPD is used asthe substrate, the reaction of the enzyme with OPD is carried out using,as a reaction buffer, a 0.05 M citrate-phosphate buffer (pH 5.0)containing 0.4 mg/mL of OPD and 0.012 to 0.02% of hydrogen peroxide, at20 to 45° C., preferably 24 to 37° C. for 1 minute to 2 hours,preferably 10 minutes to 1 hour. The reaction is stopped with a 3Nhydrochloric acid or 3M sulfuric acid in an amount one-quarter as muchas the amount of solution containing the substrate. The absorbance at490 nm is determined: (2) If TMB is used as the substrate, the reactionof the enzyme with TMB is carried out using, as a reaction buffer, a 0.1M citrate buffer (pH 5.5) containing 0.1 to 0.2 mg/mL of TMB, 0.01 to0.02% of hydrogen peroxide, 1% of DMF and 0.01% of Tween 20, at 20 to45° C., preferably 24 to 37° C. for 1 minute to 2 hours, preferably 5 to30 minutes. The reaction is stopped with a 1M phosphoric acid,hydrochloric acid or sulfuric acid in the same volume as that ofsolution containing the substrate. The absorbance at 450 nm isdetermined. (3) If the case where luminol is used as the substrate, thereaction is carried out at 20 to 37° C., preferably 24 to 37° C., for 1to 30 minutes, preferably 1 to 7 minutes. The resulting productgenerated by enzymatic reaction can be detected by a spectrophotometerif the product can be detected by its absorbance, by afluorospectrophotometer if the product can be detected by itsfluorescence, by a photometer if the product is a luminous substance,and by an electrochemical detector for detecting current generated whenthe product is oxidized on an electrode. PS2V can be detected by thepresence of the product generated by enzymatic reaction. Morespecifically, the amount of PS2V can be calculated by a calibrationcurve of the enzyme reaction product obtained by adding PS2V havingknown concentrations.

In another embodiment, the sample is mixed beforehand with the secondaryantibody and allowed to react for about an hour. A liquid containing thereaction mixture is added to wells of a reaction plate to which theprimary antibody has been immobilized and allowed to react with theprimary antibody. After B/F separation, the detection can be carried outin the same manner as described above.

PS2V can also be detected by the competitive assay. First, in the casewhere an antigen is immobilized to a solid phase, a certain amount ofPS2V is immobilized to the solid phase by a known method. PS2V used atthis time is not particularly limited so long as it includes the variantpolypeptide described above. For example, a purified PS2V, a syntheticpeptide and a hybrid protein may be used.

Subsequently, a sample is added to a reactor containing the immobilizedPS2V. A certain amount of an antibody which specifically recognizes PS2Vis added to the reactor to bring the PS2V immobilized on the solid phaseand PS2V from the sample in competition for reaction. The PS2Vimmobilized on the solid phase and the PS2V from the sample contendagainst each other for the antibody. Since the amount of the PS2Vimmobilized on the solid phase and the amount of the antibody added arefixed, the more the amount of the PS2V from the sample is, the lessantibody is bound to the PS2V immobilized on the solid phase. If a blanksample is prepared which does not contain PS2V and the amount of theantibody bound to the PS2V immobilized to the solid phase is measured,the amount of the PS2V in a non-blank sample can be obtained from adecrease in the amount of the antibody bound to the PS2V immobilized tothe solid phase. The reaction conditions such as reaction temperatureand reaction time are the same as those in the sandwich method. Afterreaction, the B/F separation is performed to remove PS2V and theantibody which are not bound to the PS2V immobilized to the solid phase.Subsequently, in the case where a labelled antibody is used as theantibody, PS2V is detected by determining the label amount. In the casewhere a biotinylated antibody is used, the antibody is conjugated usingan avidinated (or streptoavidinated) label. Alternatively, PS2V isreacted with an antibody specifically recognizing PS2V and the antibodyis conjugated with labeled antibody which recognizes the antibodyspecifically recognizing PS2V. PS2V can be detected by measuring thelabel amount. For labeling, the above-mentioned materials may be used.

Second, in the case where the antibody is immobilized to the solidphase, a certain amount of the antibody which specifically recognizesPS2V is immobilized to the solid phase by a known method. A labled PS2Vis prepared. PS2V used at this time is not particularly limited so longas it includes the variant polypeptide described above. For example, apurified PS2V, a synthetic peptide and a hybrid protein may be used.PS2V can be conjugated by a known protein labeling technique. Usablelabels are the same as described above.

Further, in a reactor in which a certain amount of the antibody whichspecifically recognizes PS2V is immobilized, a certain amount of alabeled PS2V and a sample containing an unknown amount of PS2V are fedso that the labeled PS2V and the PS2V from the sample contend againsteach other for the antibody. Since the amount of the antibodyimmobilized on the solid phase and the amount of the labeled PS2V arefixed, the more the PS2V comes from the sample, the less labeled PS2V isbound to the antibody immobilized on the solid phase. If the amount ofthe labeled PS2V bound to the antibody immobilized on the solid phase isobtained by determining a blank sample not containing PS2V, the amountof PS2V in the sample can be obtained from a decline in the amount ofthe labeled PS2V bound to the antibody immobilized on the solid phase.The reaction conditions such as the reaction temperature and thereaction time are the same as those for the sandwich method. Afterreaction, the B/F separation is conducted to remove the labeled PS2Vunbound to the solid phase. Subsequently, PS2V can be detected bydetermining the amount of the labeled PS2V captured by the solid phase.

Examples in which an antibody which specifically recognizes PS2V is usedas the primary antibody and an antibody which recognizes PS2 or PS2V isused as the secondary antibody will be hereinafter described in detail,but the present invention is not limited thereto.

Preparation of Primary Antibody (Rabbit Anti-SSMAG Antibody)

Cysteine residue was added at the N-terminal of a synthetic peptide,Gly-Arg-Leu-Ser-Ser-Met-Ala-Gly (SEQ ID NO:8). The SH group of thecysteine residue was conjugated with amino group of hemocyanin (KLH)using m-maleimidobenzoyl-N-hydroxysuccinimide ester according to theconventional method (see Biochemistry, 18, 690-697(1979)). The resultingconjugates were immunized subcutaneously in the backs of rabbits toprepare an antiserum. An antibody was purified by an affinity columncross-linked with the peptide. The antibody against the peptide wasprepared by SAWADY Technology Co., Ltd.

Preparation of Secondary Antibody (Rabbit Anti-PS2N Antibody: Anti PS2NAb)

Cysteine residue was added at the N-terminal of a synthetic peptide,Glu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu(amino acids 42nd to 58th of the amino acid sequence of PS2) (SEQ IDNO:9). The SH group of the cysteine reside was conjugated with aminogroup of hemocyanin (KLH) using m-maleimidobenzoyl-N-hydroxysuccinimideester according to the conventional method. The resulting conjugateswere immunized subcutaneously in the backs of rabbits to prepare anantiserum. An antibody was purified by an affinity column cross-linkedwith the peptide. The antibody against the peptide was prepared bySAWADY Technology Co., Ltd.

Biotinylation of Secondary Antibody

The Anti-PS2N Ab (372.1 μg/mL) prepared as described above, 1 mL, wasadded to Centricon YM30 (Cut-off=30000 Da, produced by Amicon). Theresulting mixture was centrifuged at 4500 rpm at 4° C. for 30 minutesand concentrated to 100 μL. PBS(−), 1.5 mL, was added, andcentrifugation was performed under the same conditions forconcentration. The resulting mixture was measured up to 500 μL.

The above obtained Anti-PS2N Ab solution, 500 μL, was passed through anNAP-5 column (produced by Amersham Pharmacia Biotech) equilibrated with10 mL of PBS(−). After 500 μL of eluate were disposed of, 1 mL of eluate(fraction containing the antibody) was collected. The absorbance A₂₈₀ ofthe fraction was determined (absorbance index ε=1.4 mL/mg·cm) tocalculate the concentration of the antibody (antibodyconcentration=282.4 μg/ml).

Next, a biotinylating reagent, EZ-Link Sulfo-NHS-LC-Biotin(sulfosuccinimidyl-6-(biotinamido)hexanoate, produced by PierceBiotechnology, Inc.), was added to the antibody solution in theproportion of biotinylating reagent: IgG=54 mol:1.3 mol=42:1 and allowedto react at 4° C. for 2 hours. Subsequently, the solution containing thereaction product, 1 mL, was added to Centricon YM30 and was centrifugedat 4500 rpm at 4° C. for 30 minutes and concentrated to 100 μL. TBS, 1.5mL, was added, and centrifugation and concentration were performed twiceunder the same conditions for removing the unreacted biotinylatingreagent. The absorbance A₂₈₀ was determined (absorbance index ε=1.4mL/mg·cm) to calculate the concentration of the antibody (antibodyconcentration=378.83 μg/ml).

Preparation of Genetically Recombined GST-PS2V (r GST-PS2V)

(1) Human Neuronal Cell Culture and Preparation of RNA

Human neuroblastoma SK-N—S cells (ATCC HTB-11) were cultured in an α-MEMmedium (α-Minimum Essential Medium, produced by GIBCO-BRL) containing10% v/v fetal bovine serum in a culture dish of 10 cm diameter in a CO₂incubator at 37° C. for about 48 hours up to a confluent state, then themedium was replaced with the α-MEM medium not containing the fetalbovine serum, and cultured for about 2 hours.

The culture dish was transferred into an oxygen-poor incubator (oxygenconcentration of 1% or less, 95% N₂, 5% CO₂) (produced by Coy LaboratoryProducts, Inc.), and the cultured cells were further cultured for 16hours.

The thus cultured cells were collected and washed with phosphatebuffered saline (PBS). The resulting cells were suspended in 700 μL of abuffer for cytolysis (RLT solution, produced by QIAGEN K.K.) and werecomminuted to obtain a cell extract. Total RNA was prepared from thecell extract using an RNA preparation kit (Trade name: RNeasy total RNAkit, produced by QIAGEN).

(2) Preparation of cDNA of PS2V

Using the obtained RNA (total RNA, 1 μg) as a template, single-strandedcDNA was synthesized in a 0.05 mL of a buffer (0.05M Tris-HCl, pH8.3,0.075M KCl, 0.003M MgCl₂, DTT, 0.0002M deoxynucleotides) containingoligo dT primer (50 pmole), random oligonucleotide (5 pmole) and reversetranscriptase (produced by Promega Corporation, Moloney leukaemia virusreverse transcriptase) (200 units) by one hour's reaction at 42° C.

(3) Preparation of Vector

Primers were designed at 5′ and 3′ terminals of the PS2V gene so thatthe PS2V gene could be inserted in a multi-cloning site of vector pGEX-4T-1 (produced by Amersham Pharmacia Biotech) in an in-frame state.The primers are a sense primer whose sequence is 5′-GAA TTC ATG CTC ACATTC ATG GCC TCT GAC AGC GAG GAA G-3′ (SEQ ID NO: 5) and an anti-senseprimer whose sequence is 5′-CTC GAG TCA ACC AGC CAT GGA TGA AAG CTG TCCATT CTT C-3′ (SEQ ID NO: 6). The single-stranded DNA obtained above wasused for PCR (reaction of denaturation at 95° C. for 30 seconds,reaction of elongation at 72° C. for a minute, and annealing at 55° C.for 30 seconds, 30 cycles) to prepare and isolate gene fragments. Thegene fragments were cut with restriction enzymes EcoRI and XhoI.Likewise, EcoRI and XhoI sites were cut out of p GEX-4T-1.

Next, the isolated PS2V gene fragments were purified, and were ligatedwith p GEX-4T-1 treated as described above to introduce the PS2V geneinto the multi-cloning site of p GEX-4T-1. A DNA sequencer was used toensure that the obtained product was a plasmid p GEX-4T-1/PS2V having aproper sequence. The ligation was carried out using a ligation kit(trade name: DNA Ligation Kit Ver. 2, produced by TaKaRa Bio Inc.).

(4) Expression of rGST-PS2V

The expression vector of rGST-PS2V prepared as described above wastransformed into JM109 competent cells (Escherichia coli) and culturedon an LB medium plate. PCR confirmed the introduction of the vector inemerged colonies.

Subsequently, the colonies were cultured in a liquid LB mediumcontaining 100 μg/mL of sodium ampicillin overnight.

The resulting culture liquid, 2 mL, was added to 200 mL of an LB medium.IPTG (isopropyl-β-D-thiogalactopyranoside) was added to induce theexpression of the fused protein so that the final concentration became0.1 mM after 2 hours at 37° C.

After two hours, bacterial cells were collected. The culture liquid, 50mL, was centrifuged at 8000 rpm at 4° C. for 10 minutes and deposit wascollected. The collected deposit was suspended in 3 mL of PBS containingphenylmethylsulfonyl fluoride in a final concentration of 1 mM, and theresulting suspension was ice-cooled and the bacterial cells weresonicated. After centrifugation (at 15000 rmp at 4° C. for 10 minutes),supernatant was collected.

Subsequently, the collected supernatant was added to a column of aglutathione sepharose 4B (produced by Amersham Pharmacia Biotech). Afterwashing with 50 mM Tris-HCl (pH7.4), rGST-PS2V was eluted with a 10 mMreduced glutathione solution. The absorbance of each fraction collectedwas determined, and a fraction showing a large absorbance at A₂₈₀ wassubjected to electrophoresis to confirm the presence of rGST-PS2V. Afterremoving glutathione by dialysis, the protein concentration was measuredusing Bio-Rad DC Protein Assay (produced by Bio-Rad Laboratories, Inc.).This fraction was used as rGST-PS2V standard.

EXAMPLES Example 1 Detection of rGST-PS2V by the Sandwich Method (UsingHRP and Luminal)

(1) Immobilization of Primary Antibody to Solid Phase

The primary antibody prepared as described above was added to a 96-wellplate (Cliniplate, produced by Labsystems, Inc.) by 1 pmol/well andallowed to stand at 37° C. for an hour. The 96-well plate was washedwith TBS-T (50 mM Tris-HCl 150 mM NaCl 0.05% w/v Tween 20(pH7.4))containing 0.5% w/v Block Ace (produced by Dai Nippon PharmaceuticalCo., Ltd.) by 0.32 mL/well three times. A TBS solution (50 mM Tris-HCl150 mM NaCl(pH7.4)) containing 25% w/v Block Ace was added by 300μL/well and allowed to stand at 37° C. for an hour for blocking.

(2) Determination of Chemoluminescence of rGST-PS2V

Solutions of normal serum containing 0 to 31 pmol of rGST-PS2V wereadded to the plate prepared as described above by 50 μL/well and allowedto react at 37° C. for an hour. After reaction, the plate was washedwith TBS-T containing 0.5% w/v Block Ace by 0.32 mL/well three times forB/F separation.

Subsequently, the biotinylated secondary antibody prepared as describedabove was added by 0.3 pmol/well and allowed to react at 37° C. for anhour. After reaction, the plate was washed with TBS-T containing 0.5%w/v Block Ace by 0.32 mL/well three times. A solution of TBS-Tcontaining a 5000-fold dilution of streptoavidin-HRP (produced by ZymedLaboratories, Inc.) and 0.5% w/v Block Ace was added by 50 μL/well andallowed to react at 37° C. for an hour. After reaction, the plate waswashed with TBS-T containing 0.5% w/v Block Ace by 0.32 mL/well threetimes.

Subsequently, luminol (Super Signal ELISA Pico, produced by PierceBiotechnology, Inc.), which is a luminescent substrate, was added by 80μL/well and allowed to react at 30° C. for 5 minutes. When five minutespassed from the beginning of enzymatic reaction, the accumulated amountof light emitted for 10 seconds was measured using Labsystems Luminoskan(produced by Dai Nippon Pharmaceutical Co., Ltd.). As shown in FIG. 1, aquantifiability in an amol (10⁻¹⁸ mol) order was obtained.

Measurement on SAD Patient Cerebrospinal Fluid

Measurement of PS2V was carried out on samples of 4 cases of SAD, 1 caseof subarachnoid hemorrhage and 2 cases of hydrocephalia in the samemanner as described above. FIG. 2 shows the results, which confirm thatthe measurement resulted in higher values with the SAD cases than withthe other cases and could provide an indication for diagnosis of SAD.

Determination of Absorbance of rGST-PS2V

Antigen-antibody reaction was conducted in the same manner as describedabove except that the primary antibody prepared as described above wasadded to a 96-well plate (ELISA Plate, produced by IWAKI Glass Co.) by 3pmol/well. Enzymatic reaction was started by adding TMB (produced byKPL) as a substrate by 100 μL/well and continued at 30° C. for 15minutes. Fifteen minutes after the start of the enzymatic reaction, 1MH₃PO₄ was added by 100 μL/well. The absorbance at 450 nm was determinedwith background correction in absorbance between 450 nm and 650 nm usingVERSAmax tuneable microplate reader (produced by Molecular Devices).FIG. 3 shows average absorbance. Like the determination ofchemoluminescence, a quantifiability in an amol (10⁻¹⁸ mol) order wasobtained.

Example 2 Detection of rGST-PS2V by Sandwich Method (Using ALP and pMPP(p-methoxyphenyl phosphoric acid))

(1) Immobilization of Primary Antibody to Solid Phase

The rabbit anti-SSMAG antibody (the primary antibody) prepared asdescribed above was added to a 96-well plate (Cliniplate, produced byLabsystems, Inc.) by 6.25×10⁻¹¹ mol/well and allowed to stand at 37° C.for an hour. The 96-well plate was washed with TBS-T (50 mM Tris-HCl 150mM NaCl 0.05% w/v Tween 20(pH7.4)) containing 0.1% w/v BSA (A6003,produced by SIGMA) by 300 μL/well three times. A TBS-T solutioncontaining 3% w/v BSA was added by 305 μL/well and allowed to stand at37° C. for an hour for blocking.

(2) Determination of rGST-PS2V by Electrochemical Detection

Onto the plate prepared as described above, a normal serum solutioncontaining 1×10⁻¹⁷ to 1×10⁻¹² mol of rGST-PS2V was added by 50 μL/welland allowed to react at 37° C. for an hour. After reaction, the platewas washed with TBS-T containing 0.1% w/v BSA by 300 μL/well three timesfor B/F separation.

Next, the biotinylated secondary antibody (rabbit anti-PS2N antibody)prepared as described above was added by 3.12×10⁻¹³ mol/well and allowedto react at 37° C. for an hour. After reaction, a TBS solution (50 mMTris-HCl 150 mM NaCl(pH7.4)) containing 3% w/v BSA (A6003, produced bySIGMA) was added by 305 μL/well and allowed to stand at 37° C. for anhour for blocking. After the plate was washed with TBS-T containing 0.1%w/v BSA by 0.32 mL/well three times. Then a TBS-T solution containing a5000-fold dilution of streptoavidin-ALP (produced by DAKO, Japan Co.,Ltd) and 0.5% w/v Block Ace was added by 50 μL/well and allowed to reactat 37° C. for an hour. After reaction, the plate was washed with TBS-Tcontaining 0.1% w/v BSA by 300 μL/well three times.

Next, 10 mM solution of pMPP (produced according to the method describedin Analytica Chimica Acta 393 (1999) 95-102, by Tanabe R&D) in 0.5MCB-Buffer was added as a substrate by 90 μL/well, and further, 20 μL of0.1M Mg(ClO₄)₂, 10 μL of 0.5M CB-Buffer (pH9.5) and 60 mL of RO waterwere added, followed by reaction at 37° C. for 30 minutes. Thereafter,45 μL of a 0.6M EDTA-3Na solution (adjusted to pH9.5 using NaOH) at 0°C. to stop enzymatic reaction. After stopping enzymatic reaction, themixture in each well was transferred to another plate (ELISA plate,produced by IWAKI).

p-Methoxyphenol produced by hydrolysis of pMPP by ALP was detected usinga Flow Injection Analysis-Electro Chemical-Amperometric Detection System(referred to as FIA-EC-DT hereinafter). FIG. 4 shows the construction ofthe FIA-EC-DT.

The FIA-EC-DT is composed of a degasser (Vacuum Degasser LC-27A), a pump(MICRO LC PUMP LC-100), an injection valve (produced by Reodine, 77251),an ODS column (YMC-Pack-ODS-AM, AM3C7AM 12SO₅-L502WT No. 02070458(W)), ashort column (YMC semi-micro guard cartridge column 2×10,AM12S05-0102CC) and an electrode detector.

In a fluid path of the FIA-EC-DT, 0.1 M phosphate buffer containing 45%v/v or 50% v/v methanol was passed as eluent. The degasser (VacuumDegasser LC-27A) removes air bubbles from the eluent. The pump (MICRO LCPUMP LC-100) conveys the eluent to the electrode detector at 250 μL/min.With keeping the eluent being conveyed, the injection valve (produced byReodine, 77251) was used for injection. In the present invention, 20 μLof the sample to be determined were set to flow in the fluid path of theFIA-EC-DT.

The short column was disposed between the injection valve and theelectrode detector for preventing a protein component in the sample fromreaching the electrode detector and thereby fouling the surface of theelectrode. Besides, noise current caused by the buffer and current ofcomponents for detection are separated by the short column at time toreach the detector.

FIG. 5 shows the construction of the electrode detector. Generally, theelectrochemical detection requires a working electrode (WE), a referenceelectrode (RE) and a counter electrode (CE). The working electrodeprovides an oxidation-reduction potential of a substance to be detectedand takes an oxidation-reduction current produced by oxidation-reductionreaction. The reference electrode provides a reference for setting anoxidation-reduction potential of the working electrode. The counterelectrode prevents an excess oxidation-reduction current from flowing inthe reference electrode at the oxidation-reduction reaction. Generallyused electrochemical detection systems include three-electrode systemsand four-electrode systems. The three-electrode systems have each ofthree types of the electrodes, and the four-electrode systems have twoworking electrodes and each of the other two types of the electrodes. Inthe present invention, the FIA-EC-DT electrode detector has the threeelectrode system: The reference electrode was Ag/AgCl (BAS RE-3V); thecounter electrode was integrated in a fluid path; and the workingelectrode was a glassy carbon electrode (BAS 003456) which was a radialflow-type electrode. ALS832a produced by BAS was used as anelectrochemical meter.

The substance to be detected was p-methoxyphenol, which was a hydrolyticreaction product of the substrate. Serially diluted solutions 1 nM to 1μM of p-methoxyphenol (084-01282 (special grade) by Wako Pure ChemicalIndustries, Ltd., used as it was) in 0.1 M phosphate buffer (pH6.92)were prepared, were each injected into the FIA-EC-DT and waselectrochemically detected to obtain a peak value of an oxidationcurrent and produce a calibration curve. As detection conditions, theworking electrode potential (oxidation potential) was 0.55 V vs Ag/AgCl,and the appearance time from the injection valve to the electrodedetector was 90 seconds.

Under the same detection conditions, samples according to the presentExamples after the stopping of the enzymatic reaction were injected intothe FIA-EC-DT to obtain the peak value of the oxidation current. Theresults are shown in FIGS. 6 and 7.

From the results of FIGS. 6 and 7, the dependency upon the amount ofrGST-PS2V was confirmed within the range of 1×10⁻¹⁷ mol˜1×10⁻¹² mol (10amol˜1 pmol) with reproducibility (variations) of 10% or less in themeasurement repeated three times. The signal level of 0 mol of rGST-PS2Vwas deducted as a background current value from the peak current valueof each of the samples having different numbers of rGST-PS2V molecules(0 correction). The thus confirmed dependency upon rGST-PS2V showed agood linearity within the range of 1×10⁻¹⁷ mol˜1×10⁻¹² mol (10 amol˜1pmol). This shows an amol-order detectability regarding, for example,the protein of rGST-PS2V if the electrochemical detection of ALP usingthe substrate pMPP is applied to Sandwich-ELISA.

Example 3 Determination of rGST-PS2V by Competitive Assay

(1) Immobilization of rGST-PS2V to Solid Phase

A TBS solution of 1.45 μg/mL of rGST-PS2V prepared as described abovewas added to a 96-well plate (Cliniplate, produced by Labsystems) by 50μL/well and allowed to stand at room temperature for two hours. Theplate was washed with TBS-T containing 0.1% w/v Block Ace by 0.32mL/well three times. A 50 mM Tris-HCl 150 mM NaCl solution containing25% w/v Block Ace was added by 300 μL/well and allowed to stand at 37°C. for an hour for blocking.

(2) Measurement of rGST-PS2V

Solutions of normal serum containing 0 to 31 pmol of rGST-PS2V wereadded to the plate prepared as described above by 25 μL/well.Subsequently, a 0.03 μg/mL solution of the rabbit anti-SSMAG antibodyprepared as described above (an antibody which specifically recognizesPS2V) was added by 25 μL/well and allowed to react at 37° C. for anhour. After reaction, the plate was washed with TBS-T containing 0.1%w/v Block Ace by 0.32 mL/well three times. Thereafter, a TBS-T solutionof a 1000-fold dilution of anti-rabbit IgG-HRP (produced by DAKO JapanCo., Ltd.) containing 0.1% w/v Block Ace was added by 50 μL/well andallowed to react at 37° C. for an hour.

The plate was washed with TBS-T containing 0.1% w/v Block Ace by 0.32mL/well three times. Subsequently, luminol (Super Signal ELISA Pico,produced by Pierce), which is a luminescent substrate, was added by 80μL/well and allowed to react at 30° C. for 5 minutes. When five minuteselapsed from the beginning of enzymatic reaction, the accumulated amountof light emitted for 10 seconds was measured using LabsystemsLuminoskan. FIG. 8 shows the results. It was confirmed that thecompetitive assay was also able to measure rGST-PS2V.

Example 4 Detection of rGST-PS2V by Competitive Assay

(1) Fixation of rGST-PS2V to Solid Phase

A TBS solution of 1.45 μg/mL of rGST-PS2V prepared as described abovewas fed onto a 96-hole plate (Cliniplate, produced by Labsystems) by 50μL/well and allowed to stand at room temperature for an hour. The platewas washed with TBS-T containing 0.1% w/v Block Ace by 300 μL/well threetimes. A 50 mM Tris-HCl 150 mM NaCl solution containing 25% w/v BlockAce was added by 305 μL/well and allowed to stand at 37° C. for an hourfor blocking.

(2) Detection of rGST-PS2V by Electrochemical Detection

On the plate prepared as described above, normal serum solutionscontaining 0 to 1 mol of rGST-PS2V were fed by 25 μL/well. Subsequently,0.1 mg/ml of the rabbit anti-SSMAG antibody (the primary antibody)prepared as described above was added by 25 μL/well and allowed to reactat 37° C. for an hour. The plate was washed with TBS-T containing 0.1%w/v of Block Ace by 300 μL/well three times. Thereafter, 50 μL of aTBS-T solution (containing 0.1% w/v of Block Ace) containing a4,000-fold dilution of anti-rabbit IgG-ALP (the secondary antibody(produced by DAKO) were added and allowed to react at 37° C. for anhour.

FIG. 9 shows the results of measurement. Change was recognized when theamount of the antigen rGST-PS2V in the sample exceeds 30 fmol, and it isconsidered that the competitive assay can be established with adetection sensitivity of 30 fmol.

INDUSTRIAL APPLICABILITY

According to the present invention, since PS2V can be detected with highsensitivity using body fluid samples, there can be provided test datauseful for early diagnosis of sporadic Alzheimer's disease.

The invention claimed is:
 1. A method of detecting a presenilin-2variant that has a deletion of the amino acid sequence encoded by exon 5(PS2V), said method comprising: (a) reacting a test sample with aprimary antibody, so as to form a complex between said primary antibodyand any PS2V present in said test sample, wherein the primary antibodyis immobilized on a solid phase and specifically recognizes a regionincluding the amino acid sequence of Ser-Ser-Met-Ala-Gly (SEQ ID NO:7)at the C terminal of the amino acid sequence of SEQ ID NO:2; (b-1)reacting the complex with a secondary antibody which is marked with alabel and specifically recognizes the amino acid sequence consisting ofGlu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu (SEQID NO: 9), or (b-2) reacting the complex with a secondary antibody whichis biotinylated and subsequently reacting the secondary antibody reactedcomplex with an avidinated or streptoavidinated label, wherein thesecondary antibody specifically recognizes the amino acid sequenceconsisting ofGlu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu (SEQID NO: 9), or (b-3) reacting the complex with a secondary antibody whichis biotinylated, subsequently reacting the secondary antibody reactedcomplex with avidin or streptoavidin and subsequently reacting theavidin or streptoavidin reacted complex with a biotinylated label,wherein the secondary antibody specifically recognizes the amino acidsequence consisting of Glu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu (SEQ ID NO: 9),or (b-4) reacting the complex with a secondary antibody and subsequentlyreacting the secondary antibody reacted complex with an antibody whichis marked with a label and which specifically recognizes said secondaryantibody, wherein the secondary antibody specifically recognizes theamino acid sequence consisting ofGlu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu (SEQID NO: 9); and (c) detecting the amount of label captured by the complexso as to detect said PS2V in the test sample wherein PS2V is encoded bya cDNA sequence represented by SEQ ID NO: 4 and PS2 is encoded by a cDNAsequence represented by SEQ ID NO:
 3. 2. The method of claim 1, whereinthe label in step (b-1) or (b-4) is a radio isotope, fluorescentmolecule, luminous molecule, chromophore, or ferrocene.
 3. A method ofdetecting a presenilin-2 variant that has a deletion of the amino acidsequence encoded by exon 5 (PS2V), said method comprising: (a) reactinga test sample with a primary antibody, so as to form a complex betweensaid primary antibody and any PS2V present in said test sample, whereinthe primary antibody is immobilized on a solid phase and specificallyrecognizes the amino acid sequence consisting ofGlu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu (SEQID NO: 9); (b-1) reacting the complex with a secondary antibody which ismarked with a label and specifically recognizes a region consisting ofthe amino acid sequence Ser-Ser-Met-Ala-Gly (SEQ ID NO:7) at the Cterminal of the amino acid sequence of SEQ ID NO:2, or (b-2) reactingthe complex with a secondary antibody which is biotinylated andsubsequently reacting the secondary antibody reacted complex with anavidinated or streptoavidinated label, wherein the secondary antibodyspecifically recognizes a region consisting of the amino acid sequenceSer-Ser-Met-Ala-Gly (SEQ ID NO:7) at the C terminal of the amino acidsequence of SEQ ID NO:2, or (b-3) reacting the complex with a secondaryantibody which is biotinylated, subsequently reacting the secondaryantibody reacted complex with avidin or streptoavidin and subsequentlyreacting the avidin or streptoavidin reacted complex with a biotinylatedlabel, wherein the secondary antibody specifically recognizes a regionconsisting of the amino acid sequence Ser-Ser-Met-Ala-Gly (SEQ ID NO:7)at the C-terminal of the amino acid sequence of SEQ ID NO:2, or (b-4)reacting the complex with a secondary antibody and subsequently reactingthe secondary antibody reacted complex with an antibody which is markedwith a label and which specifically recognizes said secondary antibody,wherein the secondary antibody specifically recognizes PS2V a regionconsisting of the amino acid sequence Ser-Ser-Met-Ala-Gly (SEQ ID NO:7)at the C-terminal of the amino acid sequence of SEQ ID NO:2; and (c)detecting the amount of label captured by the complex so as to detectsaid PS2V in the test sample wherein PS2V is encoded by a cDNA sequencerepresented by SEQ ID NO: 4 and PS2 is encoded by a cDNA sequencerepresented by SEQ ID NO:
 3. 4. A method of detecting a presenilin-2variant that has a deletion of the amino acid sequence encoded by exon 5(PS2V), said method comprising: (a) reacting a test sample with aprimary antibody, so as to form a complex between said primary antibodyand any PS2V present in said test sample, wherein the primary antibodyis immobilized on a solid phase and specifically recognizes PS2V aregion including the amino acid sequence of Ser-Ser-Met-Ala-Gly (SEQ IDNO:7) at the C-terminal of the amino acid sequence of SEQ ID NO:2; (b-1)reacting the complex with a secondary antibody which labeled with afirst enzyme and specifically recognizes the amino acid sequenceconsisting ofGlu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu (SEQID NO: 9), or (b-2) reacting the complex with a secondary antibody whichis biotinylated and subsequently reacting the secondary antibody reactedcomplex with an avidinated or streptoavidinated enzyme as a secondenzyme, wherein the secondary antibody specifically recognizes the aminoacid sequence consisting of SEQ ID NO: 9, or (b-3) reacting the complexwith a secondary antibody which is biotinylated and subsequentlyreacting the secondary antibody reacted complex with avidin orstreptoavidin and subsequently reacting the avidin or streptoavidinreacted complex with a biotinylated enzyme as a third enzyme, whereinthe secondary antibody specifically recognizes the amino acid sequenceconsisting of Glu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-GluAsn-Glu-Glu-Asp-Gly-Glu-Glu (SEQ ID NO: 9), or (b-4) reacting thecomplex with a secondary antibody and subsequently reacting thesecondary antibody reacted complex with an antibody which is labeledwith a fourth enzyme and specifically recognizes said secondaryantibody, wherein the secondary antibody specifically recognizes theamino acid sequence consisting of Glu-Asn-Thr-Ala-Gln-Trp-Ar-SerGln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu (SEQ ID NO: 9); and (c-1) adding afirst substrate for the first enzyme and detecting a product generatedby enzymatic reaction of the first enzyme and the first substrate, or(c-2) adding a second substrate for the second enzyme and detecting aproduct generated by enzymatic reaction of the second enzyme and thesecond substrate, or (c-3) adding a third substrate for the third enzymeand detecting a product generated by enzymatic reaction of the thirdenzyme and third substrate, or (c-4) adding a fourth substrate for thefourth enzyme and detecting a product generated by enzymatic reaction ofthe fourth enzyme and the fourth substrate wherein PS2V is encoded by acDNA sequence represented by SEQ ID NO: 4 and PS2 is encoded by a cDNAsequence represented by SEQ ID NO:
 3. 5. The method of claim 4, whereinfirst enzyme, second enzyme, third enzyme or fourth enzyme are eachindependently selected from the group consisting of horseradishperoxidase, alkaline phosphatase, β-D-galactosidase, glucose-6-phosphatedehydrogenase and luciferase.
 6. The method of claim 5, wherein thefirst enzyme, second enzyme, third enzyme or fourth enzyme are eachalkaline phosphatase, and the first substrate, second substrate, thirdsubstrate or fourth substrate are each a phosphate of a phenolderivative having an electron donative group at o-position andp-position.
 7. The method of claim 6, wherein the electron donativegroup is a lower alkoxy group.
 8. A method of detecting a presenilin-2variant that has a deletion of the amino acid sequence encoded by exon 5(PS2V), said method comprising (a) reacting a test sample with a primaryantibody, so as to form a complex between said primary antibody and anyPS2V present in said test sample, wherein the primary antibody isimmobilized on a solid phase and specifically recognizes the amino acidsequence consisting ofGlu-Asn-Thr-Ala-Gln-Trp-Arg-Ser-Gln-Glu-Asn-Glu-Glu-Asp-Gly-Glu-Glu (SEQID NO: 9); (b-1) reacting the complex with a secondary antibody which islabeled with a first enzyme and specifically recognizes regionconsisting of the amino acid sequence ef Ser-Ser-Met-Ala-Gly (SEQ IDNO:7) at the C-terminal of the amino acid sequence of SEQ ID NO:2, or(b-2) reacting the complex with a secondary antibody which isbiotinylated and subsequently reacting the secondary antibody reactedcomplex with an avidinated or streptoavidinated enzyme as a secondenzyme, wherein the secondary antibody specifically recognizes a regionconsisting of the amino acid sequence ef Ser-Ser-Met-Ala-Gly (SEQ IDNO:7) at the C-terminal of the amino acid sequence of SEQ ID NO:2, or(b-3) reacting the complex with a secondary antibody which isbiotinylated, subsequently reacting the complex with avidin orstreptoavidin and subsequently reacting the avidin or streptoavidinreacted complex with a biotinylated enzyme as a third enzyme, whereinthe secondary antibody specifically recognizes a region consisting ofthe amino acid sequence Ser-Ser-Met-Ala-Gly (SEQ ID NO:7) at theC-terminal of the amino acid sequence of SEQ ID NO:2, or (b-4) reactingthe complex with a secondary antibody and subsequently reacting thesecondary antibody complex with an antibody which is labeled with afourth enzyme and specifically recognizes said secondary antibody,wherein the secondary antibody specifically recognizes a regionconsisting of the amino acid sequence Ser-Ser-Met-Ala-Gly (SEQ ID NO:7)at the C-terminal of the amino acid sequence of SEQ ID NO: 2; and (c-1)adding a first substrate for the first enzyme and detecting a productgenerated b enzymatic reaction of the first enzyme and the firstsubstrate, or (c-2) adding a second substrate for the second enzyme anddetecting a product generated by enzymatic reaction of the second enzymeand the second substrate, or (c-3) adding a third enzyme for the thirdenzyme and detecting a product generated by enzymatic reaction of thethird enzyme and the third substrate, or (c-4) adding a fourth substratefor the fourth enzyme and detecting a product generated by enzymaticreaction of the fourth enzyme and fourth substrate wherein PS2V isencoded by a cDNA sequence represented by SEQ ID NO: 4 and PS2 is apolypeptide encoded by a cDNA sequence represented by SEQ ID NO: 3.